In general, sensitivity of emission spectrometry (emission analysis) that uses luminous phenomena of substances, materials, molecules and atoms is very high. One of known analyzing methods that use such emission is an LIF (laser-induced fluorescence) method that uses a laser beam.
The LIF method takes advantage of resonant transition of an atom and/or a molecule, which is a target to be measured. The LIF method irradiates the measurement target (atom or molecule) with the laser beam that matches an excitation level (laser beam having a tuned wavelength) to excite the measurement target. The light emission (fluorescence) takes place from the measurement target upon such excitation. The LIF method measures the light emission (fluorescence).
The density (concentration) of the measurement target is calculated from the intensity of the fluorescence, and the temperature of the measurement target is calculated from a spectral distribution of the fluorescence.
The LIF method is employed in various technical fields. In an environment technology, for example, the LIF method may be used to measure the concentration of NOx (nitrogen oxide) in the air, as disclosed in Patent Literature 1 (will be mentioned below). In metallurgy, the LIF method may be used to measure concentrations of chemical elements in a molten metal inside a metal refining furnace, as disclosed in Patent Literature 2 (will be mentioned below).
The LIF method is particularly employed in a wide range of bioanalysis in the life science technology. The florescence is useful in the bioanalysis. By detecting fluorescence radiated (emitted) from a sample S, which is irradiated with the laser beam, it is possible to extract information such as a biochemical composition of the sample S, physical characteristics of the sample S, chemical characteristics of the sample S, spectral characteristics of the sample S, a structure of a biochemical sample and a structure of an organism sample (biological sample). The duration of fluorescence (how long the fluorescence lasts) is usually in the order of ps (picosecond) to ns (nanosecond). Thus, use of the LIF method enables the measurement with the time resolution in the order of μs (microsecond), which is the in vivo action time of the biological sample.
An example of the LIF method used in the life science technology is found in Patent Literature 3 (will be mentioned below). Patent Literature 3 discloses an early warning system to terrorism in a city that uses a biological weapon such as anthrax. This early warning system employs the LIF method to detect the biological weapon.
Patent Literature 4 (will be mentioned below) discloses a measuring apparatus that causes electrophoresis of a biological sample, which is labelled (marked) with fluorescence, in a microchip and analyze the biological sample with the LIF method. Patent Literature 5 (will be mentioned below) discloses a kit that has an antibody light chain variable domain (region) polypeptide and an antibody heavy chain variable domain polypeptide, with one of the antibody light chain variable domain polypeptide and the antibody heavy chain variable domain polypeptide being labelled with a fluorescent dye. Patent Literature 5 also discloses detection of fluorescence intensity of the fluorescent pigment to measure the concentration (density) of an antigen that reacts with the kit. Patent Literature 5 shows an example in which the detection of the fluorescence intensity uses the LIF method. Patent Literature 6 (will be mentioned below) discloses an antigen measuring device that uses the LIF method.
FIG. 6 of the accompanying drawings illustrates a configuration of an antigen measuring device of Patent Literature 6 that uses the LIF method. Although the detail is not described, the measurement is carried out, for example, in the following manner. A capillary column 101 and an electrode 103a are inserted in a buffer vial 102 that holds a sample S containing an antibody, and another capillary column 101 and another electrode 103b are inserted in another buffer vial 102 that holds a sample S containing an antibody.
An antigen is introduced to the sample S, and an antibody-antigen reaction takes place in the sample. As an electric power is supplied from a high voltage source 110, the electrophoresis causes an antibody-antigen compound and the antibody to separate from the sample. Thus, the antibody-antigen compound and the antibody move through the capillary column(s) 101. In the meantime, the electric power supply is stopped, and the antibody-antigen compound and/or the antibody undergoes an enzyme reaction. Upon resuming the electric power supply, the electrophoresis causes the antibody-antigen compound and the antibody, which have finished the enzyme reaction, to move to a measuring position to which the laser beam 11 emitted from a laser device 105 is reflected by a mirror 106 and condensed by a condensing lens 104. The antibody-antigen compound and the antibody, which have finished the enzyme reaction and reached the measuring position, are irradiated with the laser beam 111 and become fluorescent (emit fluorescence).
The fluorescence passes through the mirror 106, which is designed to reflect the laser beam 111 but transmits the fluorescent light (fluorescence), and is reflected by another mirror 107, which is designed to reflect the fluorescence. Then, the fluorescence is incident to a photomultiplier tube 109 via an optical filter 108. In this manner, the intensity of the fluorescence is measured.
The optical filter 108 blocks the scattered light of the laser beam, which is excitation light, and transmits the fluorescence only. The optical filter 108 has a capability of selectively transmitting the fluorescence. The optical filter 108 prevents the scattered light of the laser beam from entering the photomultiplier tube 109. The antibody-antigen compound and the antibody, which have undergone the enzyme reaction, have different moving speeds during the electrophoresis. Thus, the fluorescence from the antibody-antigen compound that has undergone the enzyme reaction arrives at the photomultiplier tube 109 at a different timing from the fluorescence from the antibody which has undergone the enzyme reaction.